Glutaredoxins Grx3 and Grx4 regulate nuclear localisation of Aft1 and the oxidative stress response in Saccharomyces cerevisiae

Grx3 and Grx4, two monothiol glutaredoxins of Saccharomyces cerevisiae, regulate Aft1 nuclear localisation. We provide evidence of a negative regulation of Aft1 activity by Grx3 and Grx4. The Grx domain of both proteins played an important role in Aft1 translocation to the cytoplasm. This function was not, however, dependent on the availability of iron. Here we demonstrate that Grx3, Grx4 and Aft1 interact each other both in vivo and in vitro, which suggests the existence of a functional protein complex. Interestingly, each interaction occurred independently on the third member of the complex. The absence of both Grx3 and Grx4 induced a clear enrichment of G1 cells in asynchronous cultures, a slow growth phenotype, the accumulation of intracellular iron and a constitutive activation of the genes regulated by Aft1. The grx3grx4 double mutant was highly sensitive to the oxidising agents hydrogen peroxide and t-butylhydroperoxide but not to diamide. The phenotypes of the double mutant grx3grx4 characterised in this study were mainly mediated by the Aft1 function, suggesting that grx3grx4 could be a suitable cellular model for studying endogenous oxidative stress induced by deregulation of the iron homeostasis. However, our results also suggest that Grx3 and Grx4 might play additional roles in the oxidative stress response through proteins other than Aft1.

Effects of CPAP on oxidative stress and nitrate efficiency in sleep apnoea: a randomised trial.

Background: Previous studies have presented contradictory data concerning obstructive sleep apnoea syndrome (OSAS), lipid oxidation and nitric oxide (NO) bioavailability. This study was undertaken to (1) compare the concentration of 8-isoprostane and total nitrate and nitrite (NOx) in plasma of middle-aged men with OSAS and no other known co-morbidity and healthy controls of the same age, gender and body mass index; and (2) test the hypothesis that nasal continuous positive airway pressure (CPAP) therapy attenuates oxidative stress and nitrate deficiency. Methods: A prospective, randomised, placebo controlled, double-blind, crossover study was performed in 31 consecutive middle-aged men with newly diagnosed OSAS and 15 healthy control subjects. Patients with OSAS were randomised to receive sham CPAP or effective CPAP for 12 weeks. Blood pressure, urinary catecholamine levels and plasma 8-isoprostane and NOx concentrations were obtained before and after both treatment modalities. Results: Patients with OSAS had significantly higher 8-isoprostane levels (median (IQR) 42.5 (29.2-78.2) vs 20.0 (12.5-52.5) pg/ml, p = 0.041, Mann-Whitney test) and lower NOx levels (264 (165-650) vs 590 (251- 1465) mmol/l, p = 0.022) than healthy subjects. Body mass index, blood pressure and urinary catecholamines were unchanged by CPAP therapy, but 8-isoprostane concentrations decreased (38.5 (24.2-58.7) pg/ml at baseline vs 22.5 (16.2-35.3) pg/ml on CPAP, p = 0.0001) and NOx levels increased (280 (177-707) vs 1373 (981-1517) mmol/l, p = 0.0001) after CPAP. Conclusions: OSAS is associated with an increase in oxidative stress and a decrease in NOx that is normalised

Moderately oxidized oils and dietary zinc and alpha-tocopheryl acetate supplementation: effects on the oxidative stability of rabbit plasma, liver, and meat

The aim of this study was to assess the alterations in plasma, liver, and meat oxidative stability and R-tocopherol content when moderately oxidized sunflower oils were added to feeds and when feeds were supplemented with R-tocopheryl acetate (100 mg/kg) and Zn (200 mg/kg). The effects of cooking the meat and its subsequent refrigeration were also studied. When the content of primary oxidation compounds of the oil was high, rabbit plasma, liver, and meat R-tocopherol content was reduced and meat susceptibility to oxidation increased. The addition of oil with a high content of secondary oxidation compounds (oil heated at 140 'C, 31 h) to feed also led to an increase in meat susceptibi- lity to oxidation, although it presented an R-tocopherol content similar to that of nonheated oil. Feed supplementation with R-tocopheryl acetate increased tissue R-tocopherol content and improved the oxidative stability of liver and meat. However, in the latter, it was less effective when oil heated at 55 'C was added.

Oxidative Stress and Antioxidant Activity in Hypothermia and Rewarming. Can RONS Modulate the Beneficial Effects of Therapeutic Hypothermia?

Hypothermia is a condition in which core temperature drops below the level necessary to maintain bodily functions. The decrease in temperature may disrupt some physiological systems of the body, including alterations in microcirculation and reduction of oxygen supply to tissues. The lack of oxygen can induce the generation of reactive oxygen and nitrogen free radicals (RONS), followed by oxidative stress, and finally, apoptosis and/or necrosis. Furthermore, since the hypothermia is inevitably followed by a rewarming process, we should also consider its effects. Despite hypothermia and rewarming inducing injury, many benefits of hypothermia have been demonstrated when used to preserve brain, cardiac, hepatic, and intestinal function against ischemic injury. This review gives an overview of the effects of hypothermia and rewarming on the oxidant/antioxidant balance and provides hypothesis for the role of reactive oxygen species in therapeutic hypothermia.

Oxidative stress in aging: advances in proteomic approaches

Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual"s Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.

Long-term treatment with insulin induces apoptosis in brown adipocytes: role of oxidative stress

Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue.

Response of hexokinase enzymes and the insulin system to dietary carbohydrates in the common carp, Cyprinus carpio.

The response of the common carp to diets with varying amounts of digestible starch, provided either as pea meal (LP, HP, 30 and 46% peas, respectively) or as cereal (LW, HW, 30 and 46% wheat, respectively), was studied and compared with the response to a carbohydrate-free protein-rich diet (CF). Here we focused on the utilisation of dietary carbohydrates by examining the relationship between dietary starch intake, hepatic hexokinase activities, circulating insulin and muscle insulin receptor system. Plasma glucose concentration and hepatic high Km hexokinase (glucokinase, GK) activity were not affected by the content of digestible starch, but 6 h after feeding enzyme activity was higher in the fish fed carbohydrate diets. Similarly, low Km hexokinase (HK) activity was also higher in the fish 24 h after feeding. Fat gain and protein retention were significantly improved by increased digestible starch intake, especially in the HP group, which in turn, presented the highest plasma insulin levels. Glycogen stores were moderately increased by the ingestion of digestible starch. The number of insulin receptors was greater in the CF group than in fish on carbohydrates, except the HP group. Our results confirmed that the common carp uses dietary carbohydrates efficiently, especially when there are provided by peas. This efficiency might be related to the enhanced response of postprandial insulin observed in the HP group.

Catalase in fluvial biofilms: a comparison between different extraction methods and example of application in a metal-polluted river

Antioxidant enzymes are involved in important processes of cell detoxification during oxidative stress and have, therefore, been used as biomarkers in algae. Nevertheless, their limited use in fluvial biofilms may be due to the complexity of such communities. Here, a comparison between different extraction methods was performed to obtain a reliable method for catalase extraction from fluvial biofilms. Homogenization followed by glass bead disruption appeared to be the best compromise for catalase extraction. This method was then applied to a field study in a metal-polluted stream (Riou Mort, France). The most polluted sites were characterized by a catalase activity 4–6 times lower than in the low-polluted site. Results of the comparison process and its application are promising for the use of catalase activity as an early warning biomarker of toxicity using biofilms in the laboratory and in the field

Expressional regulation of key hepatic enzymes of intermediary metabolism in European seabass (Dicentrarchus labrax) during food deprivation and refeeding

We hypothesized that the analysis of mRNA level and activity of key enzymes in amino acid and carbohydrate metabolism in a feeding/fasting/refeeding setting could improve our understanding of how a carnivorous fish, like the European seabass (Dicentrarchus labrax), responds to changes in dietary intake at the hepatic level. To this end cDNA fragments encoding genes for cytosolic and mitochondrial alanine aminotransferase (cALT; mALT), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were cloned and sequenced. Measurement of mRNA levels through quantitative real-time PCR performed in livers of fasted seabass revealed a significant increase in cALT (8.5-fold induction)while promoting a drastic 45-fold down-regulation of PK in relation to the levels found in fed seabass. These observations were corroborated by enzyme activity meaning that during food deprivation an increase in the capacity of pyruvate generation happened via alanine to offset the reduction in pyruvate derived via glycolysis. After a 3-day refeeding period cALT returned to control levels while PK was not able to rebound. No alterations were detected in the expression levels of G6PDH while 6PGDH was revealed to be more sensitive specially to fasting, as confirmed by a significant 5.7-fold decrease in mRNA levels with no recovery after refeeding. Our results indicate that in early stages of refeeding, the liver prioritized the restoration of systemic normoglycemia and replenishment of hepatic glycogen. In a later stage, once regular feeding is re-established, dietary fuel may then be channeled to glycolysis and de novo lipogenesis.

Expressional regulation of key hepatic enzymes of intermediary metabolism in European seabass (Dicentrarchus labrax) during food deprivation and refeeding

We hypothesized that the analysis of mRNA level and activity of key enzymes in amino acid and carbohydrate metabolism in a feeding/fasting/refeeding setting could improve our understanding of how a carnivorous fish, like the European seabass (Dicentrarchus labrax), responds to changes in dietary intake at the hepatic level. To this end cDNA fragments encoding genes for cytosolic and mitochondrial alanine aminotransferase (cALT; mALT), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were cloned and sequenced. Measurement of mRNA levels through quantitative real-time PCR performed in livers of fasted seabass revealed a significant increase in cALT (8.5-fold induction)while promoting a drastic 45-fold down-regulation of PK in relation to the levels found in fed seabass. These observations were corroborated by enzyme activity meaning that during food deprivation an increase in the capacity of pyruvate generation happened via alanine to offset the reduction in pyruvate derived via glycolysis. After a 3-day refeeding period cALT returned to control levels while PK was not able to rebound. No alterations were detected in the expression levels of G6PDH while 6PGDH was revealed to be more sensitive specially to fasting, as confirmed by a significant 5.7-fold decrease in mRNA levels with no recovery after refeeding. Our results indicate that in early stages of refeeding, the liver prioritized the restoration of systemic normoglycemia and replenishment of hepatic glycogen. In a later stage, once regular feeding is re-established, dietary fuel may then be channeled to glycolysis and de novo lipogenesis.